Biocatalytic Transamination Process

ABSTRACT

A novel process is provided for the efficient preparation of an asymmetric compound of structural formula I: 
     
       
         
         
             
             
         
       
     
     employing dynamic kinetic resolution (DKR). The DKR process involves an enzymatic enantioselective amination reaction catalyzed by transaminases. The process can be used to manufacture key intermediates in the preparation of poly (ADP-ribose) polymerase (PARP) inhibitors which may be useful for the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/734,394, filed Dec. 7, 2012, hereby incorporated byreference herein.

REFERENCE TO SEQUENCE LISTING

A sequence listing text file is submitted via EFS-Web in compliance with37 CFR §1.52(e)(5) concurrently with the specification. The sequencelisting has the file name “23370-PCT-SEQTXT-15OCT2013”, was created onOct. 15, 2013, and is 470,397 bytes in size. The sequence listing ispart of the specification and is incorporated in its entirety byreference herein.

BACKGROUND OF THE INVENTION

This invention describes the preparation of chiral compounds byemploying dynamic kinetic resolution (DKR) involving an enzymaticenantioselective amination reaction catalyzed by a transaminase. Thisenzyme-catalyzed transaminase reaction allows convenient access tochiral compounds from an achiral starting material with highstereoselectivity.

Koszelewski et al. (2009, J. Mol. Catal. B-Enzym. 60:191-194) describespreparation of an enantiomerically enriched 4-phenylpyrrolidin-2-oneusing dynamic kinetic resolution involving an enzymatic enantioselectiveamination reaction catalyzed by ω-transaminases.

Wallace et al. (2011, Organic Process Research and Development15:831-840) describes large-scale synthesis (up to 5 kg) routes of2-{4-[(35)-Piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide relying oneither classical resolution or chiral separation.

SUMMARY OF THE INVENTION

The present invention provides an efficient process for the preparationof an asymmetric compound of formula I:

wherein:R¹ is a leaving group, a protected amino group, NO₂, or OH or itsprotected form;R² is hydrogen;R³ is (C═O)OR⁵, CH₂R⁶, or a protected aldehyde; or,R² and R³ are combined to form a nitrogen containing heterocyclylselected from

R⁴ is hydrogen or an amino protecting group;R⁵ is C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl, C₄₋₁₀ heterocyclyl, aryl, orheteroaryl; and,R⁶ is a leaving group or OH or its protected form.

The process of the present invention relates to a method for thepreparation of an asymmetric compound of formula I in an efficientenantioselective fashion via transaminase-catalyzed dynamic kineticresolution (DKR) of a compound of formula II:

wherein:R¹ is a leaving group, a protected amino group, NO₂, or OH or itsprotected form;R²′ is an aldehyde or an aldehyde equivalent; and,R³′ is (C═O)OR⁵, CH₂R⁶, or a protected aldehyde; orR^(2′) and R³′ are combined to form

The process described as part of the present invention can be used tomanufacture key intermediates in the preparation of poly (ADP-ribose)polymerase (PARP) inhibitors disclosed in U.S. Pat. No. 8,071,623, whichmay be useful for the treatment of cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides processes for the preparation of anasymmetric compound of formula I:

wherein:R¹ is a leaving group, a protected amino group, NO₂, or OH or itsprotected form;R² is hydrogen;R³ is (C═O)OR⁵, CH₂R⁶, or a protected aldehyde; or,R² and R³ are combined to form a nitrogen containing heterocyclylselected from

R⁴ is hydrogen or an amino protecting group;

R⁵ is C₁₋₆ alkyl, C₃₋₁₀ cycloalkyl, C₄₋₁₀ heterocyclyl, aryl, orheteroaryl; and,R⁶ is a leaving group or OH or its protected form;comprising a biocatalytic transamination of a compound of formula II:

wherein:R¹ is as defined above;R²′ is an aldehyde or an aldehyde equivalent; and,

R³′ is R³; or

R²′ and R³′ are combined to form

in the presence of a transaminase polypeptide, a coenzyme, and an aminodonor.

In one embodiment of the process of the present invention, R¹ is aleaving group. In a further embodiment, R¹ is Br.

In another embodiment of the process of the present invention, R² and R³are combined to form a nitrogen containing heterocyclyl selected from

and R⁴ is hydrogen. In a further embodiment, R² and R³ are combined toform

and R⁴ is hydrogen.

In a still further embodiment of the process of the present invention,R¹ is Br, R² and R³ are combined to form

and R⁴ is hydrogen.

In another embodiment of the present invention, R² is hydrogen, R³ isCH₂R⁶, R⁴ is hydrogen, and R⁶ is OH.

The present invention provides a process for the preparation of anasymmetric compound of formula III by transaminase-catalyzed DKR:

comprising a biocatalytic transamination of a compound of formula IV ora compound of formula V:

in the presence of a transaminase polypeptide, a coenzyme, and an aminodonor.

The present invention further provides a process for the preparation ofan asymmetric compound of formula VI:

by transaminase-catalyzed DKR, wherein Prot is an amino protecting groupcomprising:

(a) a biocatalytic transamination of a compound of formula IV or acompound of formula V:

-   -   in the presence of a transaminase polypeptide, a coenzyme, and        an amino donor, forming the compound of formula III:

(b) reducing the lactam of the compound of formula III, forming thecompound of formula VII:

(c) protecting the piperidine nitrogen of the compound of formula VII toform the compound of formula VI.

In one embodiment of the processes of the invention, the biocatalytictransamination provides a compound of formula I having an enantiomericexcess of at least about 95% e.e., at least about 96% e.e., at leastabout 97% e.e., at least about 98% e.e., at least about 99% e.e., or atleast about 99.9% e.e. In another embodiment of the process of thepresent invention, the transaminase-catalyzed DKR of a compound offormula II as described provides a compound of formula I having anenantiomeric excess of at least 95%. In a further embodiment of theprocess of the present invention, the transaminase-catalyzed DKR of acompound of formula II as described provides a compound of formula Ihaving an enantiomeric excess of at least 99%.

In one embodiment of the instant processes, the transaminase polypeptideis a naturally occurring transaminase. In another embodiment, thetransaminase polypeptide is a synthetic variant of a naturally occurringtransaminase. In a further embodiment, the transaminase polypeptide isselected from SEQ ID NO: 18 or SEQ ID NO: 180. In a further embodiment,the transaminase polypeptide is SEQ ID NO: 180.

In one embodiment of the instant process, isopropylamine is used as anamino donor.

In another embodiment of the instant process, pyridoxal-phosphate isused as a coenzyme.

“Amino donor” or “amine donor” refers to an amino compound which donatesan amino group to an amino acceptor, thereby becoming a carbonylspecies. Amino donors are molecules of general formula shown below,

in which each of R^(3*), R^(4*), when taken independently, is an alkyl,an alkylaryl group, or aryl group which is unsubstituted or substitutedwith one or more enzymatically non-inhibiting groups. R^(3*) can be thesame or different from R^(4*) in structure or chirality. In someembodiments, R^(3*) and R^(4*), taken together, may form a ring that isunsubstituted, substituted, or fused to other rings. Typical aminodonors that can be used with the embodiments of the present disclosureinclude chiral and achiral amino acids, and chiral and achiral amines.

“Chiral amine” refers to amines of general formula R^(α)—CH(NH₂)—R^(β)and is employed herein in its broadest sense, including a wide varietyof aliphatic and alicyclic compounds of different, and mixed, functionaltypes, characterized by the presence of a primary amino group bound to asecondary carbon atom which, in addition to a hydrogen atom, carrieseither (i) a divalent group forming a chiral cyclic structure, or (ii)two substituents (other than hydrogen) differing from each other instructure or chirality. Divalent groups forming a chiral cyclicstructure include, for example, 2-methylbutane-1,4-diyl,pentane-1,4-diyl,hexane-1,4-diyl, hexane-1,5-diyl,2-methylpentane-1,5-diyl. The two different substituents on thesecondary carbon atom (R^(α) and R^(β) above) also can vary widely andinclude alkyl, arylalkyl, aryl, halo, hydroxy, lower alkyl, loweralkyloxy, lower alkylthio, cycloalkyl, carboxy, carbalkyloxy, carbamoyl,mono- and di-(lower alkyl) substituted carbamoyl, trifluoromethyl,phenyl, nitro, amino, mono- and di-(lower alkyl) substituted amino,alkylsulfonyl, arylsulfonyl, alkylcarboxamido, arylcarboxamido, etc., aswell as alkyl, arylalkyl, or aryl substituted by the foregoing.

Exemplary amino donors that can be used with the embodiments hereininclude, by way of example and not limitation, isopropylamine (alsoreferred to as 2-aminopropane, and referred to elsewhere herein as“IPM”), α-phenethylamine (also termed 1-phenylethanamine), and itsenantiomers (S)-1-phenylethanamine and (R)-1-phenylethanamine,2-amino-4-phenylbutane, glycine, L-glutamic acid, L-glutamate,monosodium glutamate, L-alanine, D-alanine, D,L-alanine, L-asparticacid, L-lysine, D,L-ornithine, β-alanine, taurine, n-octylamine,cyclohexylamine, 1,4-butanediamine (also referred to as putrescine),1,6-hexanediamine, 6-aminohexanoic acid, 4-aminobutyric acid, tyramine,and benzyl amine, 2-aminobutane, 2-amino-1-butanol,1-amino-1-phenylethane, 1-amino-1-(2-methoxy-5-fluorophenyl)ethane,1-amino-1-phenylpropane, 1-amino-1-(4-hydroxyphenyl)propane,1-amino-1-(4-bromophenyl)propane, 1-amino-1-(4-nitrophenyl)propane,1-phenyl-2-aminopropane, 1-(3-trifluoromethylphenyl)-2-aminopropane,2-aminopropanol, 1-amino-1-phenylbutane, 1-phenyl-2-aminobutane,1-(2,5-dimethoxy-4-methylphenyl)-2-aminobutane, 1-phenyl-3-aminobutane,1-(4-hydroxyphenyl)-3-aminobutane, 1-amino-2-methylcyclopentane,1-amino-3-methylcyclopentane, 1-amino-2-methylcyclohexane,1-amino-1-(2-naphthyl)ethane, 3-methylcyclopentylamine,2-methylcyclopentylamine, 2-ethylcyclopentylamine,2-methylcyclohexylamine, 3-methylcyclohexylamine, 1-aminotetralin,2-aminotetralin, 2-amino-5-methoxytetralin, and 1-aminoindan, includingboth (R) and (S) single isomers where possible and including allpossible salts of the amines.

“Amino acceptor” and “amine acceptor,” “keto substrate,” “keto,” and“ketone” are used interchangeably herein to refer to a carbonyl (keto,or ketone) compound which accepts an amino group from a donor amine.Amino acceptors are molecules of general formula shown below,

in which each of R^(1*), R^(2*), when taken independently, is an alkyl,an alkylaryl group, or aryl group which is unsubstituted or substitutedwith one or more enzymatically acceptable groups. R^(1*) may be the sameor different from R^(2*) in structure or chirality. In some embodiments,R^(1*) and R^(2*), taken together, may form a ring that isunsubstituted, substituted, or fused to other rings. Amino acceptorsinclude keto carboxylic acids, alkanones (ketones), and alkanals(aldehydes).

“Coenzyme,” as used herein, refers to a non-protein compound thatoperates in combination with an enzyme in catalyzing a reaction. As usedherein, “coenzyme” is intended to encompass the vitamin B₆ familycompounds PLP, PN, PL, PM, PNP, and PMP.

“Pyridoxal-phosphate,” “PLP,” “pyridoxal-5′-phosphate,” “PYP,” and “P5P”are used interchangeably herein to refer to a compound that acts as acoenzyme in transaminase reactions. In some embodiments, pyridoxalphosphate is defined by the structure1-(4′-formyl-3′-hydroxy-2′-methyl-5′-pyridyl)methoxyphosphonic acid, CASnumber [54-47-7]. Pyridoxal-5′-phosphate can be produced in vivo byphosphorylation and oxidation of pyridoxol (also known as Vitamin B₆).In transamination reactions using transaminase enzymes, the amine groupof the amino donor is transferred to the coenzyme to produce a ketoby-product, while pyridoxal-5′-phosphate is converted to pyridoxaminephosphate. Pyridoxal-5′-phosphate is regenerated by reaction with adifferent keto compound (the amino acceptor). The transfer of the aminegroup from pyridoxamine phosphate to the amino acceptor produces anamine and regenerates the coenzyme. In some embodiments, thepyridoxal-5′-phosphate can be replaced by other members of the vitaminB₆ family, including pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM),and their phosphorylated counterparts; pyridoxine phosphate (PNP), andpyridoxamine phosphate (PMP).

“Leaving group” is defined as a term that would be understood by one ofordinary skill in the art; that is, a group on a carbon where, uponreaction, a new bond is to be formed and the carbon loses the group uponformation of the new bond. A typical example employing a suitableleaving group is a nucleophilic substitution reaction, e.g., on a sp³hybridized carbon (S_(N)2 or S_(N)1), e.g. where the leaving group is ahalide, such as a bromide, the reactant might be benzyl bromide. Anothertypical example of such a reaction is a nucleophilic aromaticsubstitution reaction (SNAr). Another example is an insertion reaction(for example by a transition metal) into the bond between an aromaticreaction partner bearing a leaving group followed by reductive coupling.“Leaving group” is not limited to such mechanistic restrictions.Examples of suitable leaving groups include halogens (fluorine,chlorine, bromine or iodine), optionally substituted aryl or alkylsulfonates, phosphonates, azides and —S(O)₀₋₂R where R is, for exampleoptionally substituted alkyl, optionally substituted aryl, or optionallysubstituted heteroaryl. Those of skill in the art of organic synthesiswill readily identify suitable leaving groups to perform a desiredreaction under different reaction conditions. Non-limitingcharacteristics and examples of leaving groups can be found, for examplein Organic Chemistry, 2nd ed., Francis Carey (1992), pages 328-331;Introduction to Organic Chemistry, 2d ed., Andrew Streitwieser andClayton Heathcock (1981), pages 169-171; and Organic Chemistry, 5th Ed.,John McMurry, Brooks/Cole Publishing (2000), pages 398 and 408; all ofwhich are incorporated herein by reference.

“Protecting group” refers to a group of atoms that mask, reduce orprevent the reactivity of the functional group when attached to areactive functional group in a molecule. Typically, a protecting groupmay be selectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Wuts and Greene, “Greene'sProtective Groups in Organic Synthesis,” 4^(th) Ed., Wiley Interscience(2006), and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Functional groups that canhave a protecting group include, but are not limited to, hydroxy, amino,and carboxy groups.

Representative amino protecting groups include, but are not limited to,formyl, acetyl (Ac), trifluoroacetyl, benzyl (Bn), benzoyl (Bz),carbamate, benzyloxycarbonyl (“CBZ”), p-methoxybenzyl carbonyl (Moz orMeOZ), tert-butoxycarbonyl (“Boc”), trimethylsilyl (“TMS”),2-trimethylsilyl-ethanesulfonyl (“SES”), trityl and substituted tritylgroups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”),nitro-veratryloxycarbonyl (“NVOC”), p-methoxybenzyl (PMB), tosyl (Ts)and the like.

Representative hydroxyl protecting groups include, but are not limitedto, those where the hydroxyl group is either acylated (e.g., methyl andethyl esters, acetate or propionate groups or glycol esters) oralkylated such as benzyl and trityl ethers, as well as alkyl ethers,tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPSgroups) and allyl ethers. Other protecting groups can be found in thereferences noted herein.

A “protected aldehyde” is defined as the term would be understood by oneof ordinary skill in the art; that is, the aldehyde is protected with agroup such that it may be converted under assays conditions to anunprotected aldehyde. Examples of protected aldehydes include, but arenot limited to, an acetal or hemiacetal which can be converted into afree aldehyde group by treatment with acids (organic or inorganicacids), such as acetal groups formed with a polyalcohol such as propanediol or ethylene glycol, or hemiacetal groups in a sugar or in asugar-related compound such as an aldose sugar, e.g. glucose orgalactose. Further examples of protected aldehydes are imino groups(e.g., ═NH groups), which give aldehyde groups upon treatment withacids; thioacetal or dithioacetal groups (e.g., C(SR)₂ groups wherein Rmay be an alkyl radical), which give aldehyde groups upon treatment withmercury salts; oxime groups (e.g., ═NOH groups), which give aldehydegroups upon treatment with acids; hydrazone groups (e.g., ═N—NHR groupswherein R may be an alkyl radical), which give aldehyde groups upontreatment with acids; and imidazolone or imidazolidine groups orbenzothiazole or dihydrobenzothiazole groups, which give aldehydes uponhydrolysis, e.g. with acid.

As used herein except where noted, “alkyl” is intended to include bothbranched- and straight-chain saturated aliphatic hydrocarbon groupshaving the specified number of carbon atoms. For example, “C₁-C₆” or“C₁₋₆,” as in “C₁-C₆ alkyl” or “C₁₋₆ alkyl,” is defined to includegroups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branchedarrangement. C₁₋₆ alkyl includes all of the hexyl alkyl and pentyl alkylisomers as well as n-, iso-, sec- and t-butyl, n- and isopropyl, ethyland methyl. As another example, C₁₋₄ alkyl means n-, iso-, sec- andt-butyl, n- and isopropyl, ethyl and methyl. As another example, C₁-C₁₀alkyl specifically includes methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on.If no number is specified, 1-10 carbon atoms are intended for linear orbranched alkyl groups. Commonly used abbreviations for alkyl groups areused throughout the specification, e.g. methyl may be represented byconventional abbreviations including “Me” or CH₃ or a symbol that is anextended bond without defined terminal group, e.g.

ethyl may be represented by “Et” or CH₂CH₃, propyl may be represented by“Pr” or CH₂CH₂CH₃, butyl may be represented by “Bu” or CH₂CH₂CH₂CH₃,etc. The term “cycloalkyl” means a monocyclic saturated aliphatichydrocarbon group having the specified number of carbon atoms. Forexample, “cycloalkyl” includes cyclopropyl, methyl-cyclopropyl,2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, cyclopentenyl,cyclobutenyl and so on.

“Aryl,” unless otherwise indicated, is intended to mean any stablemonocyclic or bicyclic carbon ring of up to 7 atoms in each ring,wherein at least one ring is aromatic. Examples of such aryl elementsinclude phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl. Incases where the aryl substituent is bicyclic and one ring isnon-aromatic, it is understood that attachment is via the aromatic ring.In an embodiment, aryl is phenyl.

The term “heteroaryl,” as used herein, represents a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and contains from 1 to 4 heteroatoms selected from the groupconsisting of O, N and S. Heteroaryl groups within the scope of thisdefinition include but are not limited to: acridinyl, carbazolyl,cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl,thienyl, benzothienyl, benzofuranyl, benzimidazolonyl, benzoxazolonyl,quinolinyl, isoquinolinyl, dihydroisoindolonyl, imidazopyridinyl,isoindolonyl, indazolyl, oxazolyl, oxadiazolyl, isoxazolyl, indolyl,pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl,tetrahydroquinoline. As with the definition of heterocycle below,“heteroaryl” is also understood to include the N-oxide derivative of anynitrogen-containing heteroaryl. In cases where the heteroarylsubstituent is bicyclic and one ring is non-aromatic or contains noheteroatoms, it is understood that attachment is via the aromatic ringor via the heteroatom containing ring, respectively.

The term “heterocycle” or “heterocyclyl,” as used herein, is intended tomean a 3- to 10-membered aromatic or nonaromatic heterocycle containingfrom 1 to 4 heteroatoms selected from the group consisting of O, N andS, and includes bicyclic groups. For the purposes of this invention, theterm “heterocyclic” is also considered to be synonymous with the terms“heterocycle” and “heterocyclyl” and is understood as also having thedefinitions set forth herein. “Heterocyclyl” therefore includes theabove mentioned heteroaryls, as well as dihydro and tetrahydro analogsthereof. Further examples of “heterocyclyl” include, but are not limitedto the following: azetidinyl, benzoimidazolyl, benzofuranyl,benzofurazanyl, benzopyrazolyl, benzotriazolyl, benzothiophenyl,benzoxazolyl, carbazolyl, carbolinyl, cinnolinyl, furanyl, imidazolyl,indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthpyridinyl, oxadiazolyl,oxooxazolidinyl, oxazolyl, oxazoline, oxopiperazinyl, oxopyrrolidinyl,oxomorpholinyl, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl,pyridazinyl, pyridopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl,quinazolinyl, quinolyl, quinoxalinyl, tetrahydropyranyl,tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydroisoquinolinyl,tetrazolyl, tetrazolopyridyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl,pyridin-2-onyl, pyrrolidinyl, morpholinyl, thiomorpholinyl,dihydrobenzoimidazolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl,dihydrobenzoxazolyl, dihydrofuranyl, dihydroimidazolyl, dihydroindolyl,dihydroisooxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl,dihydrooxazolyl, dihydropyrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,dioxidothiomorpholinyl, methylenedioxybenzoyl, tetrahydrofuranyl, andtetrahydrothienyl, and N-oxides thereof. Attachment of a heterocyclylsubstituent can occur via a carbon atom or via a heteroatom.

“Protein,” “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated there fromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Where amixture contains more than two diastereomers it is common to report theratio of diastereomers or “diastereomeric ratio” rather thandiastereomeric excess. Enantiomeric excess and diastereomeric excess aretypes of stereomeric excess. “High stereoselectivity,” in reference tothe process of the present invention, refers the capability ofconverting a substrate to the asymmetric amine product with at leastabout 85% stereomeric excess.

“Transaminase,” “transaminase polypeptide” and “transaminase enzyme,” asused interchangeably herein, refer to a polypeptide having an enzymaticcapability of transferring an amino group (NH₂), a pair of electrons,and a proton from a primary amine of an amino donor to a carbonyl group(C═O; i.e., a keto group) of an amino acceptor molecule. Transaminasesas used herein include naturally occurring (wild-type) transaminases, aswell as non-naturally occurring engineered polypeptides generated byhuman manipulation. In an embodiment of the instant process, atransaminase polypeptide having the amino acid sequence as set forth inSEQ ID NO: 18 or SEQ ID NO: 180 is used. In an embodiment of the instantprocess, a transaminase polypeptide having a polynucleotide sequence asset forth in SEQ ID NO: 17 or SEQ ID NO: 179 is used.

Transaminase Polypeptides

Transaminases have been identified from various organisms, such asAlcaligenes denitrificans, Arthrobacter, Bordetella bronchiseptica,Bordetella parapertussis, Brucella melitensis, Burkholderia malle,Burkholderia pseudomallei, Chromobacterium violaceum, Oceanicolagranulosus HTCC2516, Oceanobacter sp. RED65, Oceanospirillum sp. MED92,Pseudomonas putida, Ralstonia solanacearum, Rhizobium meliloti,Rhizobium sp. (strain NGR234), Bacillus thuringensis, Vibrio fluvialisand Klebsiella pneumoniae (see, e.g., Shin et al., 2001, Biosci.Biotechnol. Biochem. 65: 1782-1788). Both R-selective and S-selectivetransaminases are known. The wild-type transaminase from Arthrobactersp. KNK168 is an R-selective, pyridoxal 5′-phosphate (PLP)-dependentenzyme that produces R-amines from some substrates (see, e.g., Iwasakiet al., 2006, Appl. Microbiol. Biotechnol., 69:499-505; U.S. Pat. No.7,169,592).

U.S. application Ser. No. 12/714,397, filed Feb. 26, 2010 (published asUS20100285541), and PCT International application serial no.PCT/US2010/025685, filed Feb. 26, 2010 (published as WO 2010/099501),disclose engineered transaminase polypeptides derived from the naturallyoccurring transaminase of Arthrobacter sp. KNK168. These transaminasepolypeptides have increased stability to temperature and/or organicsolvent and have been adapted to have enzymatic activity towardsstructurally different amino acceptor molecules (see also, e.g., Savileet al., 2010, Science 329(5989):305-9). PCT International patentapplication serial no. PCT/US2011/046932, filed Aug. 8, 2011 (publishedas WO 2012/024104), further describes non-naturally occurringtransaminase polypeptides derived from Arthrobacter sp. KNK168engineered to have improved properties, such as increasedstereoselectivity. These synthetic variants of the naturally occurringArthrobacter sp. KNK168 transaminase comprise amino acid sequences thathave one or more residue differences as compared to the wild-typesequence. For example, the residue differences may occur at residuepositions that affect one or more functional properties of the enzyme,including but not limited to stereoselectivity, substrate and/or productbinding (e.g., resistance to substrate and/or product inhibition),activity (e.g., percent conversion of substrate to product),thermostability, solvent stability, expression, or various combinationsthereof.

In one embodiment of the present invention, the asymmetric synthesis ofcompounds of formula I from compounds of formula II via biocatalytictransamination utilizes a naturally occurring transaminase polypeptide.In another embodiment, the synthesis reaction of the invention utilizesa synthetic variant of a naturally occurring transaminase. In a furtherembodiment, the synthetic, engineered transaminase polypeptide isderived from a transaminase of Arthrobacter sp. KNK168, including theArthrobacter sp. KNK168 transaminase polypeptide as set forth in SEQ IDNO: 2, wherein the synthetic variant comprises an amino acid sequencehaving one or more residue differences as compared to the wild-typesequence of SEQ ID NO: 2.

In a further embodiment, the transaminase polypeptide comprises orconsists of an engineered transaminase polypeptide as described in PCTInternational application serial no. PCT/US2011/046932, filed Aug. 8,2011 (published as WO 2012/024104), which is hereby incorporated byreference herein, having among other things a high stereoselectivity.These engineered polypeptides are non-naturally occurring transaminasesengineered to have improved properties, such as increasedstereoselectivity, increased activity, increased thermostability, andtolerance of increased substrate and/or product concentration (e.g.,decreased product inhibition).

As described in detail in PCT International application serial no.PCT/US2011/046932, engineered transaminases were previously identifiedby optimizing the reported wild-type omega transaminase polypeptide fromArthrobacter sp. KNK168 of SEQ ID NO: 2. First, a transaminasepolypeptide derived from SEQ ID NO: 2 having a single amino acid changerelative to the wild-type sequence (I306V) was generated having theamino acid sequence as set forth in SEQ ID NO: 4 (encoded by thepolynucleotide sequence as set forth in SEQ ID NO: 3). The syntheticpolynucleotide of SEQ ID NO: 3 was optimized for increased expressionand thermostability by inserting active and silent mutations which aredescribed in U.S. application Ser. No. 12/714,397, filed Feb. 26, 2010,which is incorporated herein by reference. This optimization resulted inthe synthetic polynucleotide of SEQ ID NO: 5, which encodes theengineered polypeptide of SEQ ID NO: 6, having the following 24 aminoacid differences relative to the naturally occurring transaminase ofArthrobacter sp. KNK168 (SEQ ID NO: 2): S8P; Y60F; L61Y; H62T; V65A;V69T; D81G; M94I; I96L; F1221; G136F; A169L; V1991; A209L; G215C; G217N;S223P; L269P; L273Y; T282S; A284G; P297S; 1306V; and S321P. Theengineered transaminase polypeptide of SEQ ID NO: 6 was used as thestarting backbone for further optimization to generate polynucleotidesencoding additional engineered transaminase polypeptides (seePCT/US2011/046932; supra).

Therefore, in a further embodiment of the present invention, theasymmetric synthesis of compounds of formula I from compounds of formulaII via biocatalytic transamination utilizes a transaminase polypeptidethat is a synthetic variant of a naturally occurring transaminase andcomprises the acid sequence as set forth in SEQ ID NO: 6.

In another embodiment of the instant process, the transaminasepolypeptide comprises an amino acid sequence having at least 80%sequence identity (e.g., at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to thereference polypeptide of SEQ ID NO: 6.

In a further embodiment of the present invention, the transaminasepolypeptide used in a process of the invention comprises an amino acidsequence having at least 80% sequence identity to the referencepolypeptide of SEQ ID NO: 6 and an amino acid residue difference ascompared to SEQ ID NO: 6 at one or more of the following positions: X2;X4; X5; X7; X8; X9; X10; X11; X14; X22; X28; X37; X38; X41; X42; X44;X52; X54; X55; X56; X58; X69; X94; X99; X108; X124; X126; X135; X136;X141; X142; X150; X155; X156; X157; X164; X165; X171; X182; X199; X209;X210; X213; X215; X217; X218; X223; X245; X257; X265; X267; X296; andX328. In some embodiments of the instant process, the amino acid residuedifferences of the transaminase polypeptide as compared to SEQ ID NO: 6are selected from the following: X2K; X2Q; X2S; X4I; X4L; X5H; X5I; X5L;X5N; X5S; X5T; X5V; X7A; X8T; X9N; X9Q; X9S; X10V; X11K; X14R; X22I;X28P; X37R; X38G; X41F; X42A; X44Q; X44V; X52K; X54K; X54N; X54P; X54R;X55L; X56G; X56L; X56S; X58L; X69C; X69V; X69W; X94L; X99L; X108V;X124F; X1241; X124L; X124R; X124V; X126A; X126T; X135Q; X136W; X141L;X142R; X142T; X150A; X150F; X150N; X155A; X156A; X156F; X156G; X1565;X156T; X157L; X164A; X165N; X171A; X182T; X199F; X199R; X199Y; X209C;X209D; X209E; X2105; X213P; X215F; X215Y; X2175; X218M; X223I; X223L;X223M; X223N; X2455; X257F; X265T; X267V; X2965; and X328I.

In some embodiments of the instant process, the transaminase polypeptidecomprises one or more combinations of amino acid differences as comparedto SEQ ID NO: 6 selected from the following: (a) X124V and X2105; (b)X124V, X136W and X2105; (c) X69V and X136W; (d) X69V and X215Y; (e) X69Vand X2175; (f) X69V, X1241 and X136W; (g) X69V, X136W and X257F; (h)X44V and X223N; (i) X56S, X69V, X136W and X265T; and (j) X28P, X69V andX136W.

Exemplary engineered polypeptides having various combinations of aminoacid differences resulting in improved properties are provided in thesequence listing incorporated by reference herein and include thepolypeptides as set forth in SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, and206. Exemplary polynucleotide sequences encoding these transaminases areprovided in the sequence listing incorporated by reference herein andinclude the polynucleotides as set forth in SEQ ID NO: 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173,175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,203, and 205.

Thus, in another embodiment, a transaminase polypeptide utilized in theprocess of the present invention comprises the amino acid sequence asset forth in SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, or 206.

In a further embodiment of the instant process, a transaminasepolypeptide having an amino acid sequence as set forth in SEQ ID NO: 18is used. SEQ ID NO: 18 has the following amino acid differences relativeto SEQ ID NO: 6: T69V, S1241 and F136W In a further embodiment of theinstant process, a transaminase polypeptide having a polynucleotidesequence as set forth in SEQ ID NO: 17 is used.

In a further embodiment of the instant process, a transaminasepolypeptide having an amino acid sequence as set forth in SEQ ID NO: 180is used. SEQ ID NO: 180 has the following amino acid differencesrelative to SEQ ID NO: 6: A2S, ASH, T69V, S1241, F136W and C215F.

In a further embodiment of the instant process, a transaminasepolypeptide having a polynucleotide sequence as set forth in SEQ ID NO:179 is used.

In addition to the exemplary engineered polypeptides described herein,the process of the present disclosure can be carried out usingengineered transaminase polypeptides having improved enzymaticproperties (e.g., as disclosed above) and comprising furthermodifications of the amino acid sequence. Such engineered polypeptidescan be derived from the exemplary polypeptides and have amino acidsequences retaining some percent identity to the exemplary engineeredpolypeptides and one or more of the amino acid differences relative toSEQ ID NO: 6 that are associated with the improved enzymatic property.Techniques and methods for deriving further engineered polypeptides areknown in the art and include the methods of directed evolution asdescribed herein. For example, any of the exemplary engineeredpolypeptides can be used as the starting amino acid sequence (i.e., the“backbone” sequence) for subsequent rounds of evolution in which alibrary of genes encoding additional amino acid differences in thebackbone is synthesized, expressed, and screened in high-throughput forparticular improved properties (e.g., thermostability, total substrateconversion, stereoselectivity, etc.). The design of the libraries can becontrolled such that only certain amino acid positions are allowed tochange, while others are not. Thus, a backbone set of amino aciddifferences that are associated with improved properties can bemaintained throughout the directed evolution process. The most improvedengineered polypeptides from each round could then be used as the parent“backbone” sequence for subsequent rounds of evolution. The resultingengineered transaminase polypeptides, having further improvements in itsproperties, will retain some or all of the starting backbone amino aciddifferences and include new amino acid differences, typically whileretaining an overall sequence identity to the starting backbone of atleast 80%. It is contemplated, however, that one or more of the backboneamino acid differences can be changed during the directed evolutionprocess leading to further improved properties in the engineeredpolypeptides. Further improvements at later rounds of evolution such as“fine tuning” an engineered polypeptide for certain process conditions(e.g., solvent conditions/concentrations, increased substrate and/orcoenzyme loading, pH, and temperature changes) may be generated byincluding amino acid differences at positions that had been maintainedas unchanged throughout earlier rounds of evolution.

In some embodiments, the engineered transaminase polypeptides useful inthe process of the instant invention comprise an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to a reference amino acid sequenceselected from any one of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, and 206. Theamino acid sequence can include one or more residue differences ascompared to SEQ ID NO:6 at the following residue positions: X2; X4; X5;X7; X8; X9; X10; X11; X14; X22; X28; X37; X38; X41; X42; X44; X52; X54;X55; X56; X58; X69; X94; X99; X108; X124; X126; X135; X136; X141; X142;X150; X155; X156; X157; X164; X165; X171; X182; X199; X209; X210; X213;X215; X217; X218; X223; X245; X257; X265; X267; X296; and X328. Theamino acid sequence can include one or more residue differences ascompared to SEQ ID NO:6 selected from the following: X2K; X2Q; X2S; X4I;X4L; X5H; X5I; X5L; X5N; X5S; X5T; X5V; X7A; X8T; X9N; X9Q; X9S; X10V;X11K; X14R; X22I; X28P; X37R; X38G; X41F; X42A; X44Q; X44V; X52K; X54K;X54N; X54P; X54R; X55L; X56G; X56L; X565; X58L; X69C; X69V; X69W; X94L;X99L; X108V; X124F; X1241; X124L; X124R; X124V; X126A; X126T; X135Q;X136W; X141L; X142R; X142T; X150A; X150F; X150N; X155A; X156A; X156F;X156G; X1565; X156T; X157L; X164A; X165N; X171A; X182T; X199F; X199R;X199Y; X209C; X209D; X209E; X2105; X213P; X215F; X215Y; X2175; X218M;X223I; X223L; X223M; X223N; X2455; X257F; X265T; X267V; X2965; andX328I.

In some embodiments, the engineered transaminase polypeptides useful inthe process of the instant invention comprise an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to a reference amino acid sequenceselected from any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, and 206; andfurther comprises one or more combinations of amino acid differences ascompared to SEQ ID NO: 6 selected from the following: (a) X124V andX2105; (b) X124V, X136W and X2105; (c) X69V and X136W; (d) X69V andX215Y; (e) X69V and X2175; (f) X69V, X1241 and X136W; (g) X69V, X136Wand X257F; (h) X44V and X223N; (i) X56S, X69V, X136W and X265T; and (j)X28P, X69V and X136W. In addition to one or more of the abovecombinations, the engineered polypeptide amino acid sequence can furthercomprise one or more amino acid residue differences as compared to SEQID NO: 6 selected from the following: X2K; X2Q; X2S; X4I; X4L; X5H; X5I;X5L; X5N; X5S; X5T; X5V; X54K; X54N; X54P; X54R; X56G; X94L; X1241;X126A; X126T; X150A; X150N; X1565; X215F; and X267V.

In some embodiments, the engineered transaminase polypeptides useful inthe process of the instant invention comprise an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity to a reference amino acid sequenceselected from any one of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, and 206;comprises an amino acid difference as compared to SEQ ID NO: 6 at one ormore of the following positions: X28; X69; X124; X126; X136; X150; X156;X199; X209; X215; X217; and X223; and further comprises an amino aciddifference as compared to SEQ ID NO: 6 at one or more of the followingpositions: X2; X4; X5; X7; X8; X9; X10; X11; X14; X22; X37; X38; X41;X42; X44; X52; X54; X55; X56; X58; X94; X99; X108; X126; X135; X141;X142; X155; X157; X164; X165; X171; X182; X210; X213; X218; X245; X257;X265; X267; X296; and X328. In some embodiments, the amino aciddifferences as compared to SEQ ID NO: 6 at positions X28; X69; X124;X126; X136; X150; X156; X199; X209; X215; X217; and/or X223, areselected from the following: X28P; X69C; X69V; X69W; X124F; X1241;X124L; X124R; X124V; X126A; X126T; X136W; X150A; X150N; X1565; X199F;X199R; X199Y; X209C; X209D; X209E; X215F; X215Y; X2175; X223I; X223L;X223M; and X223N. In other embodiments, the amino acid differences ofthe transaminase polypeptide as compared to SEQ ID NO: 6 at positionsX28; X69; X124; X126; X136; X150; X156; X199; X209; X215; X217; and/orX223 are selected from the following: X28P; X69C; X136W; X150N; X1565;X199F; X199Y; and X2175. In some embodiments of the process, the aminoacid differences of the transaminase polypeptide as compared to SEQ IDNO: 6 at positions X2; X4; X5; X7; X8; X9; X10; X11; X14; X22; X37; X38;X41; X42; X44; X52; X54; X55; X56; X58; X94; X99; X108; X135; X141;X142; X155; X157; X164; X165; X171; X182; X210; X213; X218; X245; X257;X265; X267; X296; and X328 are selected from: X2K; X2Q; X2S; X4I; X4L;X5H; X5I; X5L; X5N; X5S; X5T; X5V; X7A; X8T; X9N; X9Q; X9S; X10V; X11K;X14R; X22I; X37R; X38G; X41F; X42A; X44Q; X44V; X52K; X54K; X54N; X54P;X54R; X55L; X56G; X56L; X56S; X58L; X94L; X99L; X108V; X135Q; X141L;X142R; X142T; X155A; X156A; X156F; X156G; X1565; X156T; X157L; X164A;X165N; X171A; X182T; X2105; X213P; X218M; X2455; X257F; X265T; X267V;X2965; and X328I.

In some embodiments, the instant process uses a non-naturally occurringpolypeptide comprising an amino acid sequence having at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, and 206; and furthercomprises the set of one or more amino acid residue differences ascompared to SEQ ID NO:6 found in any one of SEQ ID NO: 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, and 206.

Amino acid residue differences at other positions relative to thewild-type sequence of SEQ ID NO: 2 and the affect of these differenceson enzyme function are provide by other engineered transaminasepolypeptides disclosed in U.S. application Ser. No. 12/714,397, filedFeb. 26, 2010. One or more of the amino acid differences as compared tothe wild-type sequence of SEQ ID NO: 2, provided in the engineeredtransaminase polypeptide amino acid sequences of U.S. application Ser.No. 12/714,397, filed Feb. 26, 2010 (see e.g., Table 2 of U.S.application Ser. No. 12/714,397), could also be introduced into aengineered transaminase polypeptide of the present disclosure.

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine HIS H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleotides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated. In addition, the following terms aredefined as:

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence that can be isolated from a sourcein nature and which has not been intentionally modified by humanmanipulation.

“Derived from” as used herein in the context of engineered transaminaseenzymes, identifies the originating transaminase enzyme, and/or the geneencoding such transaminase enzyme, upon which the engineering was based.For example, the engineered transaminase enzyme of SEQ ID NO:34 wasobtained by artificially evolving, over multiple generations the geneencoding the Arthrobacter sp. KNK168 transaminase enzyme of SEQ ID NO:2.Thus, this engineered transaminase enzyme is “derived from” thewild-type transaminase of SEQ ID NO:2.

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present disclosure. Each control sequence maybe native or foreign to the nucleic acid sequence encoding thepolypeptide. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Recombinant” or “engineered” or “non-naturally occurring” when usedwith reference to, e.g., a cell, nucleic acid, or polypeptide, refers toa material, or a material corresponding to the natural or native form ofthe material, that has been modified in a manner that would nototherwise exist in nature, or is identical thereto but produced orderived from synthetic materials and/or by manipulation usingrecombinant techniques. Non-limiting examples include, among others,recombinant cells expressing genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence for optimal alignment of the two sequences. Thepercentage may be calculated by determining the number of positions atwhich the identical nucleic acid base or amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Alternatively, the percentage may becalculated by determining the number of positions at which either theidentical nucleic acid base or amino acid residue occurs in bothsequences or a nucleic acid base or amino acid residue is aligned with agap to yield the number of matched positions, dividing the number ofmatched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Those of skill in the art appreciate that there aremany established algorithms available to align two sequences. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch,1970, J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package), or by visualinspection (see generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1995Supplement) (Ausubel)). Examples of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, NucleicAcids Res. 3389-3402, respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915).Exemplary determination of sequence alignment and % sequence identitycan employ the BESTFIT or GAP programs in the GCG Wisconsin Softwarepackage (Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. A “reference sequence” can be based on aprimary amino acid sequence, where the reference sequence is a sequencethat can have one or more changes in the primary sequence. For instance,a “reference sequence based on SEQ ID NO:2 having at the residuecorresponding to X9 a threonine” refers to a reference sequence in whichthe corresponding amino acid residue at X9 in SEQ ID NO:2, which is analanine, has been changed to threonine.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Corresponding to,” “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredtransaminase, can be aligned to a reference sequence by introducing gapsto optimize residue matches between the two sequences. In these cases,although the gaps are present, the numbering of the residue in the givenamino acid or polynucleotide sequence is made with respect to thereference sequence to which it has been aligned.

“Amino acid difference” or “residue difference” refers to a change inthe amino acid residue at a position of a polypeptide sequence relativeto the amino acid residue at a corresponding position in a referencesequence. The positions of amino acid differences generally are referredto herein as “Xn,” where n refers to the corresponding position in thereference sequence upon which the residue difference is based. Forexample, a “residue difference at position X3 as compared to SEQ ID NO:2” refers to a change of the amino acid residue at the polypeptideposition corresponding to position 3 of SEQ ID NO:2. Thus, if thereference polypeptide of SEQ ID NO: 2 has a glutamine at position 3,then a “residue difference at position X3 as compared to SEQ ID NO:2” isan amino acid substitution of any residue other than glutamine at theposition of the polypeptide corresponding to position 3 of SEQ ID NO: 2.In most instances herein, the specific amino acid residue difference ata position is indicated as “XnY” where “Xn” specified the correspondingposition as described above, and “Y” is the single letter identifier ofthe amino acid found in the engineered polypeptide (i.e., the differentresidue than in the reference polypeptide). In some instances, thepresent disclosure also provides specific amino acid differences denotedby the conventional notation “AnB”, where A is the single letteridentifier of the residue in the reference sequence, “n” is the numberof the residue position in the reference sequence, and B is the singleletter identifier of the residue substitution in the sequence of theengineered polypeptide. In some instances, a polypeptide can include oneor more amino acid residue differences relative to a reference sequence,which is indicated by a list of the specified positions where changesare made relative to the reference sequence. The present process may useengineered polypeptide sequences which comprise one or more amino aciddifferences that include either/or both conservative andnon-conservative amino acid substitutions.

“Conservative amino acid substitution” refers to a substitution of aresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid, e.g., alanine, valine, leucine, and isoleucine; an amino acid withhydroxyl side chain is substituted with another amino acid with ahydroxyl side chain, e.g., serine and threonine; an amino acids havingaromatic side chains is substituted with another amino acid having anaromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, andhistidine; an amino acid with a basic side chain is substituted withanother amino acid with a basis side chain, e.g., lysine and arginine;an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain, e.g., aspartic acid or glutamicacid; and a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively. Exemplaryconservative substitutions are provided in Table 1 below:

TABLE 1 Residue Possible Conservative Substitutions A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) N, Q, S, T Other polar H, Y, W, F Other aromatic (H, Y, W, F) C,P None

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

In some embodiments, the present process uses engineered transaminasepolypeptides that comprise a polypeptide fragment of any of theengineered transaminase polypeptides described herein that retains thefunctional activity and/or improved property of that engineeredtransaminase. A polypeptide fragment that may be capable of asymmetricsynthesis of compounds of formula I from compounds of formula II viabiocatalytic transamination include fragments comprising at least about80%, 90%, 95%, 98%, or 99% of a full-length amino acid sequence of anexemplary engineered transaminase polypeptide of SEQ ID NO: 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, and 206.

In some embodiments, the process of the instant invention uses anengineered transaminase polypeptide having an amino acid sequencecomprising a deletion as compared to any one of the engineeredtransaminase polypeptides described herein, such as the exemplaryengineered polypeptides of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, and 206.Thus, for each and every embodiment of the engineered transaminasepolypeptides of the present process, the amino acid sequence cancomprise deletions of one or more amino acids, 2 or more amino acids, 3or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 ormore amino acids, 8 or more amino acids, 10 or more amino acids, 15 ormore amino acids, or 20 or more amino acids, up to 10% of the totalnumber of amino acids, up to 20% of the total number of amino acids, orup to 30% of the total number of amino acids of the transaminasepolypeptides, where the associated functional activity and/or improvedproperties of the engineered transaminase is maintained. The deletionscan comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 amino acid residues. The number of deletions can be 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 30, 30, 35, 40, 45, 50, 55, or 60 amino acids. In some embodiments,the deletions can comprise deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, 25 or 30 amino acidresidues.

In some embodiments, the process uses an engineered transaminasepolypeptide having an amino acid sequence comprising an insertion ascompared to any one of the engineered transaminase polypeptidesdescribed herein, such as the exemplary engineered polypeptides of SEQID NO: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,194, 196, 198, 200, 202, 204, and 206. Thus, for each embodiment of thetransaminase polypeptides utilized in the instant process, theinsertions can comprise one or more amino acids, 2 or more amino acids,3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6or more amino acids, 8 or more amino acids, 10 or more amino acids, 15or more amino acids, or 20 or more amino acids, where the associatedfunctional activity and/or improved properties of the engineeredtransaminase described herein is maintained. The insertions can be toamino or carboxy terminus of the transaminase, or internal portions ofthe transaminase polypeptide.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered transaminase enzyme or polypeptide.Deletions can be directed to the internal portions and/or terminalportions of the polypeptide. The deletion can comprise a continuoussegment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. The improvedengineered transaminase enzymes comprise insertions of one or more aminoacids to the naturally occurring transaminase polypeptide as well asinsertions of one or more amino acids to other improved transaminasepolypeptides. Insertions can be in the internal portions of thepolypeptide, or to the carboxy or amino terminus. Insertions as usedherein include fusion proteins as is known in the art. The insertion canbe a contiguous segment of amino acids or separated by one or more ofthe amino acids in the naturally occurring polypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length transaminase polypeptide,for example the polypeptide of SEQ ID NO:2 or an engineeredtransaminase.

In some embodiments, the transaminase polypeptides of the instantprocess can be in the form of fusion polypeptides in which theengineered polypeptides are fused to other polypeptides, such as, by wayof example and not limitation, antibody tags (e.g., myc epitope),purification sequences (e.g., His-tags for binding to metals), and celllocalization signals (e.g., secretion signals). Thus, the engineeredpolypeptides described herein can be used with or without fusions toother polypeptides.

The engineered transaminase polypeptides described herein are notrestricted to the genetically encoded amino acids. In addition to thegenetically encoded amino acids, the polypeptides described herein maybe comprised, either in whole or in part, of naturally-occurring and/orsynthetic non-encoded amino acids. Certain commonly encounterednon-encoded amino acids of which the polypeptides described herein maybe comprised include, but are not limited to: the D-stereomers of thegenetically-encoded amino acids; 2,3-diaminopropionic acid (Dpr);α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovalericacid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine(Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug);N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine(Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine(Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisolencine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid. Asdescribed above the various modifications introduced into the naturallyoccurring polypeptide to generate an engineered transaminase enzyme canbe targeted to a specific property of the enzyme.

In some embodiments, the transaminase polypeptides used in the instantprocess are bound to a substrate. The transaminase polypeptide can bebound non-covalently or covalently. Various methods for conjugation tosubstrates, e.g., membranes, beads, glass, etc. are described in, amongothers, Hermanson, G. T., Bioconjugate Techniques, Second Edition,Academic Press; (2008), and Bioconjugation Protocols: Strategies andMethods, In Methods in Molecular Biology, C. M. Niemeyer ed., HumanaPress (2004); the disclosures of which are incorporated herein byreference.

The polynucleotides encoding the exemplary engineered transaminasesuseful in the present process are selected from SEQ ID NO: 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199,201, 203, and 205. These polynucleotides may be manipulated in a varietyof ways well-known in the art to provide for expression of theengineered polypeptides, including further sequence alteration bycodon-optimization to improve expression, insertion in a suitableexpression with or without further control sequences, and transformationinto a host cell suitable for expression and production of thepolypeptide, as further described in detail in PCT Internationalapplication serial no. PCT/US2011/046932, supra.

To make the transaminase polynucleotides and polypeptides for use in thepresent process, the naturally-occurring transaminase enzyme thatcatalyzes the transamination reaction can be obtained (or derived) fromArthrobacter sp. KNK168. In some embodiments, the parent polynucleotidesequence is codon optimized to enhance expression of the transaminase ina specified host cell. The parental polynucleotide sequence encoding thewild-type polypeptide of Arthrobacter sp. KNK168 has been described (seee.g., Iwasaki et al., Appl. Microbiol. Biotechnol., 2006, 69: 499-505).Preparation of engineered transaminases based on this parental sequenceare also described in U.S. application Ser. No. 12/714,397, filed Feb.26, 2010 and International application PCT/US2010/025685, filed Feb. 26,2010.

The engineered transaminases can be obtained by subjecting thepolynucleotide encoding the naturally occurring transaminase tomutagenesis and/or directed evolution methods, as discussed above. Anexemplary directed evolution technique is mutagenesis and/or DNAshuffling as described in Stemmer, 1994, Proc Natl Acad Sci USA91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746. Other directedevolution procedures that can be used include, among others, staggeredextension process (StEP), in vitro recombination (Zhao et al., 1998,Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al., 1994, PCRMethods Appl. 3:S136-S140), and cassette mutagenesis (Black et al.,1996, Proc Natl Acad Sci USA 93:3525-3529). Mutagenesis and directedevolution techniques useful for the purposes herein are also describedin e.g., Ling, et al., 1997, Anal. Biochem. 254(2):157-78; Dale et al.,1996, Methods Mol. Biol. 57:369-74; Smith, 1985, Ann. Rev. Genet.19:423-462; Botstein et al., 1985, Science 229:1193-1201; Carter, 1986,Biochem. J. 237:1-7; Kramer et al., 1984, Cell, 38:879-887; Wells etal., 1985, Gene 34:315-323; Minshull et al., 1999, Curr Opin Chem Biol3:284-290; Christians et al., 1999, Nature Biotech 17:259-264; Crameriet al., 1998, Nature 391:288-291; Crameri et al., 1997, Nature Biotech15:436-438; Zhang et al., 1997, Proc Natl Acad Sci USA 94:45-4-4509;Crameri et al., 1996, Nature Biotech 14:315-319; Stemmer, 1994, Nature370:389-391; Stemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; PCTPubl. Nos. WO 95/22625, WO 97/0078, WO 97/35966, WO 98/27230, WO00/42651, and WO 01/75767; and U.S. Pat. No. 6,537,746. All publicationsand patent are hereby incorporated by reference herein.

The clones obtained following mutagenesis treatment can be screened forengineered transaminases having a desired improved enzyme property.Measuring enzyme activity from the expression libraries can be performedusing the standard biochemistry techniques, such as HPLC analysisfollowing OPA derivatization of the product amine (see, e.g.,PCT/US2011/046932; supra).

Where the improved enzyme property desired is thermostability, enzymeactivity may be measured after subjecting the enzyme preparations to adefined temperature and measuring the amount of enzyme activityremaining after heat treatments. Clones containing a polynucleotideencoding a transaminase are then isolated, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell.

“Improved enzyme property” refers to a transaminase polypeptide thatexhibits an improvement in any enzyme property as compared to areference transaminase. For the engineered transaminase polypeptides,the comparison is generally made to the wild-type transaminase enzyme,although in some embodiments, the reference transaminase can be anotherimproved engineered transaminase. Enzyme properties for whichimprovement is desirable include, but are not limited to, enzymaticactivity (which can be expressed in terms of percent conversion of thesubstrate), thermostability, solvent stability, pH activity profile,coenzyme requirements, refractoriness to inhibitors (e.g., substrate orproduct inhibition), stereospecificity, and stereoselectivity (includingenantioselectivity).

“Increased enzymatic activity” refers to an improved property of theengineered transaminase polypeptides, which can be represented by anincrease in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount oftransaminase) as compared to the reference transaminase enzyme. Anyproperty relating to enzyme activity may be affected, including theclassical enzyme properties of K_(m), V_(max) or k_(cat), changes ofwhich can lead to increased enzymatic activity. Improvements in enzymeactivity can be from about 1.1 times the enzymatic activity of thecorresponding wild-type transaminase enzyme, to as much as 2 times, 5times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, ormore enzymatic activity than the naturally occurring transaminase oranother engineered transaminase from which the transaminase polypeptideswere derived. It is understood by the skilled artisan that the activityof any enzyme is diffusion limited such that the catalytic turnover ratecannot exceed the diffusion rate of the substrate, including anyrequired coenzymes. The theoretical maximum of the diffusion limit, ork_(cat)/K_(m), is generally about 10⁸ to 10⁹ (M⁻¹ s⁻¹). Hence, anyimprovements in the enzyme activity of the transaminase will have anupper limit related to the diffusion rate of the substrates acted on bythe transaminase enzyme.

Transaminase activity can be measured by any one of standard assays,such as by monitoring changes in spectrophotometric properties ofreactants or products. The amount of products produced can be measuredby High-Performance Liquid Chromatography (HPLC) separation combinedwith UV absorbance or fluorescent detection following o-phthaldialdehyde(OPA) derivatization. Comparisons of enzyme activities are made using adefined preparation of enzyme, a defined assay under a set condition,and one or more defined substrates, as further described in detailherein. Generally, when lysates are compared, the numbers of cells andthe amount of protein assayed are determined as well as use of identicalexpression systems and identical host cells to minimize variations inamount of enzyme produced by the host cells and present in the lysates.

“Conversion” refers to the enzymatic conversion of the substrate(s) tothe corresponding product(s). “Percent conversion” refers to the percentof the substrate that is converted to the product within a period oftime under specified conditions. Thus, the “enzymatic activity” or“activity” of a transaminase polypeptide can be expressed as “percentconversion” of the substrate to the product.

“Thermostable” refers to a transaminase polypeptide that maintainssimilar activity (more than 60% to 80% for example) after exposure toelevated temperatures (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the wild-type enzyme.

“Solvent stable” refers to a transaminase polypeptide that maintainssimilar activity (more than e.g., 60% to 80%) after exposure to varyingconcentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol,dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran,acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for aperiod of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme.

“Thermo- and solvent stable” refers to a transaminase polypeptide thatis both thermostable and solvent stable.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of thedisclosure can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., 1981, TetLett 22:1859-69, or the method described by Matthes et al., 1984, EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors. In addition, essentially anynucleic acid can be obtained from any of a variety of commercialsources.

Reaction Conditions

As described further below, and illustrated in the Examples, the presentprocess contemplates ranges of suitable reaction conditions that can beused in the enzymatic process disclosed, including but not limited toranges of pH, temperature, buffer, solvent system, substrate loading,transaminase polypeptide loading, coenzyme loading, atmosphere, andreaction time. Further suitable reaction conditions for carrying out themethod for the transaminase-catalyzed DKR of a compound formula II toform an asymmetric compound of formula I can be readily optimized byroutine experimentation that includes, but is not limited to, contactinga transaminase polypeptide and substrate (compound of formula II) underexperimental reaction conditions of concentration, pH, temperature,solvent conditions, and detecting the production of the compound offormula I, for example, using the methods described in the Examplesprovided herein.

“Suitable reaction conditions” refers to those conditions in thebiocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, coenzyme loading, temperature, pH, buffers,co-solvents, etc.) under which a transaminase polypeptide is capable ofconverting a compound of formula II to a compound of formula I.Exemplary “suitable reaction conditions” are provided in the presentdisclosure and illustrated by the Examples.

“Substrate” in the context of a biocatalyst mediated process refers tothe compound or molecule acted on by the biocatalyst. For example, anexemplary substrate for the transaminase biocatalyst in the processesdisclosed herein is a compound of formula II.

The use of a lower concentration of transaminase polypeptide in aprocess of the invention may reduce the amount of residual protein thatmay need to be removed in subsequent steps for purification of thecompound of formula I. In some embodiments of the process, the suitablereaction conditions comprise a transaminase polypeptide concentration ofabout 0.1 to about 15 g/L, about 0.5 to about 10 g/L, about 1.0 to about5 g/L, about 2 to about 5 g/L, about 15 g/L, about 10 g/L, about 5, g/L,about 3 g/L, about 2 g/L, about 1.5 g/L, about 1.0 g/L, about 0.75 g/L,or even lower concentration.

In some embodiments of the process, the amino donor is isopropylamine(also referred to herein as “IPM” or “iPrNH₂”), putrescine, L-lysine,α-phenethylamine, D-alanine, L-alanine, or D,L-alanine, orD,L-ornithine. In some embodiments, the amino donor is IPM, putrescine,L-lysine, D- or L-alanine. In some embodiments, the amino donor is IPM.In some embodiments, the suitable reaction conditions comprise the aminodonor at a concentration of at least about 0.5 M, at least about 1.0 M,at least about 2.5 M, at least about 5.0 M, at least about 7.5 M, atleast about 10.0 M, or more.

Suitable reaction conditions for the process of the instant inventionrequire a coenzyme. Engineered transaminases, as disclosed herein, mayrequire far less coenzyme than reactions catalyzed with wild-typetransaminase enzymes. Coenzymes useful in the disclosed methods include,but are not limited to, pyridoxal-5′-phosphate (also known aspyridoxal-phosphate, PLP, P5P). In some embodiments, the coenzyme is amember of the vitamin B6 family, selected from PLP, pyridoxine (PN),pyridoxal (PL), pyridoxamine (PM), and their phosphorylatedcounterparts; pyridoxine phosphate (PNP), and pyridoxamine phosphate(PMP). In some embodiments, the coenzyme is PLP. In some embodiments,the coenzyme is present naturally in the cell extract and does not needto be supplemented. In some embodiments of the methods, the suitablereaction conditions comprise coenzyme added to the enzyme reactionmixture. In some embodiments, the coenzyme is added either at thebeginning of the reaction and/or additional coenzyme is added during thereaction.

In some embodiments of the process, the suitable reaction conditions canfurther comprise the presence of the reduced coenzyme, nicotinamideadenine dinucleotide (NADH), which can act to limit the inactivation ofthe transaminase enzyme (see, e.g., van Ophem et al., 1998, Biochemistry37(9):2879-88). In such embodiments where NADH is present, a coenzymeregeneration system, such as glucose dehydrogenase (GDH) and glucose orformate dehydrogenase and formate can be used to regenerate the NADH inthe reaction medium.

In some embodiments of the process, the suitable reaction conditionscomprise a substrate compound of formula II loading of at least about 5g/L, at least about 10 g/L, at least about 15 g/L, at least about 20g/L, at least about 30 g/L, at least about 50 g/L, at least about 75g/L, at least about 100 g/L, or even greater.

In certain embodiments of the process, the temperature of the suitablereaction conditions can be chosen to maximize the reaction rate athigher temperatures while maintaining the activity of the enzyme forsufficient duration for efficient synthesis. Where higher temperaturesare used, polypeptides with increased thermostability can be selected tocarry out the process. For example, the engineered polypeptides of thepresent disclosure have increased thermal stability relative tonaturally occurring transaminase polypeptide e.g., the wild typepolypeptide of SEQ ID NO: 2. In some embodiments of the method thesuitable reaction conditions comprise a temperature of between about 25°C. and about 75° C., between about 35° C. and about 65° C., betweenabout 40° C. and about 60° C., at least about 30° C., at least about 35°C., at least about 40° C., at least about 45° C., or at least about 50°C., or about 60° C., or more. In certain embodiments, the temperatureduring the enzymatic reaction can be maintained at a temperaturethroughout the course of the reaction. In some embodiments, thetemperature during the enzymatic reaction can be adjusted over atemperature profile during the course of the reaction.

The methods for preparing compounds of formula I of the presentdisclosure are generally carried out in a solvent. Suitable solventsinclude water, aqueous buffer solutions, organic solvents, and/orco-solvent systems, which generally comprise aqueous solvents andorganic solvents. The aqueous solvent (water or aqueous co-solventsystem) may be pH-buffered or unbuffered.

In certain embodiments, the process for preparing compounds of formula Iof the present invention can be carried out with the pH of the reactionmixture and may be maintained at a desired pH or within a desired pHrange by the addition of an acid or a base during the course of thereaction. In certain embodiments of the process, the pH of the reactionmixture may be allowed to change, or be changed during the course of thereaction. Thus, it is contemplated that in some embodiments the pH maybe controlled by using an aqueous solvent that comprises a buffer. Insome embodiments of the method, the suitable reaction conditionscomprise a solution pH of between about pH 8.5 and about pH 11.5,between about pH 9.0 and about pH 11.5, between about pH 9.5 and aboutpH 11.0, at least about pH 8.5, at least about pH 9.0, at least about pH9.5, at least about pH 10.0, or at least about pH 10.5. Suitable buffersto maintain desired pH ranges are known in the art and include, forexample, phosphate buffer, triethanolamine buffer, and the like.Combinations of buffering and acid or base addition may also be used. Insome embodiments, the buffer is TEA (e.g., about 0.025 M to about 0.25 MTEA). In some embodiments of the process the suitable reactionconditions comprise a buffer solution of about 0.05 M borate to about0.25 M borate, or about 0.1 M borate. In some embodiments, the reactionconditions comprise water as a suitable solvent with no buffer present.

In some embodiments, the process for preparing compounds of formula Iare generally carried out in an aqueous co-solvent system comprising anorganic solvent (e.g., ethanol, isopropanol (IPA), dimethyl sulfoxide(DMSO), ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methylt-butyl ether (MTBE), toluene, and the like), ionic liquids (e.g.,1-ethyl 4-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium tetrafluoroborate,1-butyl-3-methylimidazolium hexafluorophosphate, and the like). Theorganic solvent component of an aqueous co-solvent system may bemiscible with the aqueous component, providing a single liquid phase, ormay be partly miscible or immiscible with the aqueous component,providing two liquid phases. Exemplary aqueous co-solvent systemscomprises water and one or more organic solvent. In general, an organicsolvent component of an aqueous co-solvent system is selected such thatit does not completely inactivate the transaminase enzyme. Appropriateco-solvent systems can be readily identified by measuring the enzymaticactivity of the specified engineered transaminase enzyme with a definedsubstrate of interest in the candidate solvent system, utilizing anenzyme activity assay, such as those described herein. In someembodiments of the process, the suitable reaction conditions comprise anaqueous co-solvent comprising DMSO at a concentration of at least about5% (v/v), at least about 10% (v/v), at least about 20% (v/v), at leastabout 30% (v/v), or at least about 40% (v/v).

In carrying out the transamination reactions described in the process ofthe instant invention, the transaminase polypeptide may be added to thereaction mixture in the form of a purified enzyme, whole cellstransformed with gene(s) encoding the enzyme, and/or as cell extractsand/or lysates of such cells. Whole cells transformed with gene(s)encoding the transaminase enzyme or cell extracts, lysates thereof, andisolated enzymes may be employed in a variety of different forms,including solid (e.g., lyophilized, spray-dried, and the like) orsemisolid (e.g., a crude paste). The cell extracts or cell lysates maybe partially purified by precipitation (ammonium sulfate,polyethyleneimine, heat treatment or the like, followed by a desaltingprocedure prior to lyophilization (e.g., ultrafiltration, dialysis, andthe like). Any of the cell preparations may be stabilized bycrosslinking using known crosslinking agents, such as, for example,glutaraldehyde or immobilization to a solid phase (e.g., Eupergit C, andthe like). In some embodiments where the transaminase polypeptide can beexpressed in the form of a secreted polypeptide and the culture mediumcontaining the secreted polypeptides can be used in the disclosedmethod.

In some embodiments, solid reactants (e.g., enzyme, salts, etc.) may beprovided to the reaction in a variety of different forms, includingpowder (e.g., lyophilized, spray dried, and the like), solution,emulsion, suspension, and the like. The reactants can be readilylyophilized or spray dried using methods and equipment that are known tothose having ordinary skill in the art. For example, the proteinsolution can be frozen at −80° C. in small aliquots, then added to apre-chilled lyophilization chamber, followed by the application of avacuum.

In some embodiments, the order of addition of reactants is not critical.The reactants may be added together at the same time to a solvent (e.g.,monophasic solvent, biphasic aqueous co-solvent system, and the like),or alternatively, some of the reactants may be added separately, andsome together at different time points. For example, the coenzyme,transaminase, and transaminase substrate may be added first to thesolvent. For improved mixing efficiency when an aqueous co-solventsystem is used, the transaminase, and coenzyme may be added and mixedinto the aqueous phase first. The organic phase may then be added andmixed in, followed by addition of the transaminase substrate.Alternatively, the transaminase substrate may be premixed in the organicphase, prior to addition to the aqueous phase.

The quantities of reactants used in the transamination reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of transaminase substrate employed. Thosehaving ordinary skill in the art will readily understand how to varythese quantities to tailor them to the desired level of productivity andscale of production. Transformation of substrate to product can bemonitored using known methods by detecting substrate and/or product.Suitable methods include gas chromatography, HPLC, and the like.

In some embodiments, the process can further comprise a step of removalof the carbonyl by-product formed from the amino group donor when theamino group is transferred to the transaminase substrate. Such removalin situ can reduce the rate of the reverse reaction such that theforward reaction dominates and more substrate is then converted toproduct. Removal of the carbonyl by-product can be carried in a numberof ways. Where the amino group donor is an amino acid, such as alanine,the carbonyl by-product, a keto acid, can be removed by reaction with aperoxide (see, e.g., US 2008/0213845, incorporated herein by reference).Peroxides which can be used include, among others, hydrogen peroxide;peroxyacids (peracids) such as peracetic acid (CH₃CO₃H),trifluoroperacetic acid and metachloroperoxybenzoic acid; organicperoxides such as t-butyl peroxide ((CH₃)₃COOH), or other selectiveoxidants such as tetrapropylammonium perruthenate, MnO₂, KMnO₄,ruthenium tetroxide and related compounds. Alternatively, pyruvateremoval can be achieved via its reduction to lactate by employinglactate dehydrogenase to shift equilibrium to the product amine (see,e.g., Koszelewski et al., 2008, Adv. Syn. Catal. 350:2761-2766).Pyruvate removal can also be achieved via its decarboxylation to carbondioxide acetaldehyde by employing pyruvate decarboxylase (see, e.g.,Hohne et al., 2008, Chem BioChem 9:363-365).

In some embodiments of the process, where the choice of the amino donorresults in a carbonyl by-product that has a vapor pressure higher thanwater (e.g., a low boiling co-product such as a volatile organiccarbonyl compound), the carbonyl by-product can be removed by spargingthe reaction solution with a non-reactive gas or by applying a vacuum tolower the reaction pressure and removing the carbonyl by-product presentin the gas phase. A non-reactive gas is any gas that does not react withthe reaction components. Various non-reactive gases include nitrogen andnoble gases (e.g., inert gases). In some embodiments, the non-reactivegas is nitrogen gas.

In some embodiments, the amino donor used in the process isisopropylamine (IPM), which forms the carbonyl by-product acetone upontransfer of the amino group to the amino group acceptor. The acetone canbe removed by sparging with nitrogen gas at elevated temperatures orapplying a vacuum to the reaction solution and removing the acetone fromthe gas phase by an acetone trap, such as a condenser or other coldtrap. Alternatively, the acetone can be removed by reduction toisopropanol using a ketoreductase. A nitrogen sweep can also be used toprevent formation of a ketone side product, ensuring that the reactionremains inert at all times.

In some embodiments of the process where the carbonyl by-product isremoved, the corresponding amino group donor can be added during thetransamination reaction to replenish the amino group donor and/ormaintain the pH of the reaction. Replenishing the amino group donor alsoshifts the equilibrium towards product formation, thereby increasing theconversion of substrate to product. Thus, in some embodiments whereinthe amino group donor is IPM and the acetone product is removed in situ,the method can further comprise a step of adding IPM to the reactionsolution to replenish the amino group donor lost during the acetoneremoval and to maintain the pH of the reaction (e.g., at about 8.5).

Alternatively, in embodiments where an amino acid is used as amino groupdonor, the keto acid carbonyl by-product can be recycled back to theamino acid by reaction with ammonia and NADH using an appropriate aminoacid dehydrogenase enzyme, thereby replenishing the amino group donor.

In some embodiments, the process of the instant invention can furthercomprise extraction, isolation, purification, and/or crystallization ofthe compound of formula I, each of which can be carried out under arange of conditions.

These and other aspects of the invention will be apparent from theteachings contained herein.

EXAMPLES

Examples provided are intended to assist in a further understanding ofthe invention. Particular materials employed, species and conditions areintended to be illustrative of the invention and not limiting of thereasonable scope thereof.

Certain starting materials and reagents are either commerciallyavailable or known in the chemical scientific or patent literature.Purification procedures include, for example, distillation,crystallization, and normal or reverse phase high performance liquidchromatography.

The abbreviations used herein have the following tabulated meanings (seeTable 2). Abbreviations not tabulated below have their meanings ascommonly used unless specifically stated otherwise.

TABLE 2 NaOH = Sodium hydroxide AlCl₃ = Aluminum chloride H₂SO₄ =Sulfuric acid IPA = Isopropyl alcohol Na₂CO₃ = Sodium carbonate MgSO₄ =Magnesium sulfate Me₃SOI = Trimethyl sulfoxonium iodide KOt-Bu_((s)) =Potassium tert-butoxide DMSO = Dimethyl sulfoxide THF = TetrahydrofuranNa₂SO₄ = Sodium sulfate ZnBr₂ = Zinc bromide NaHSO₃ = Sodium bisulfitePhMe = Toluene NaCl = Sodium chloride iPrNH₂ = Isopropylamine MeCN =Acetonitrile PLP = Pyridoxal-phosphate NaBH₄ = Sodium borohydride EtOH =Ethanol BF₃•THF = Boron trifluoride tetrahydrofuran complex MeOH =Methanol NH₄OH = Ammonium hydroxide LCAP = Liquid chromatography areapercent MeLi = Methyllithium DBU = 1,8-Diazabicyclo[5.4.0]undec-7-eneDIBAL = Diisobutylaluminium hydride MsCl = Methanesulfonyl chloride Et₃N= Triethylamine CH₂Cl₂ = Dichloromethane DMF = Dimethylformamide

Example 1

The following Example 1 describes synthesis of the chiral lactam 7 usingDKR transamination:

1.1 Acylation

A mixture of succinic anhydride 1 (110 g) and bromobenzene (695 mL) wascooled to 2-5° C. then added AlCl₃ (294 g). The slurry was allowed towarm to RT and then aged until the reaction was complete judged by HPLC.The reaction mixture was then transferred slowly into a cold HClsolution resulting in the formation of a white precipitate. The whiteslurry was filtered through a fitted funnel rinsing with H₂O. To theoff-white product was added MTBE and extracted with aq. NaOH. Theaqueous layer was cooled in an ice bath. Concentrated HCl was added dropwise to adjust the solution pH to 1, resulting in the formation of awhite slurry. The slurry was collected on a fitted funnel, rinsed withH₂O, and dried under vacuum with a N₂ sweep at RT to give the targetcompound (265 g, 93% corrected yield) as a white powder.

1.2 Esterification

A mixture of the acid 2 (205 g), IPA (4 L) and conc. H₂SO₄ (2.13 mL/3.91g) was heated to a gentle reflux until the reaction was complete judgedby HPLC. The solution was then cooled to RT and concentrated to a volumeof 350-400 mL. The residue was dissolved in MTBE (1.2 L), washed withaq. Na₂CO₃ followed by water. After dried over MgSO₄, the filtrate wassolvent-switched into heptane. The slurry was then filtered, and thecake was washed with cold heptane. After drying under vacuum, the targetcompound (223.5 g, 93% corrected yield) was obtained as a white powder.

1.3 Epoxidation

A mixture of Me₃SOI (230 g) and DMSO (300 mL) was added KOt-Bu (113 g)followed by DMSO (300 mL). The mixture was aged for a further 1.5 hr. Ina separate flask, ketone 3 (230 g) was dissolved in a mixture of THF(250 mL) and DMSO (150 mL), and the resulting solution was added dropwise to the glide solution. The mixture was aged for 2 hr at RT, addedhexanes (1 L), and then quenched by the addition of ice-water (600 mL).The layers were cut, and the organic layer was washed with water thenwith brine. The slightly cloudy yellow organic layer was dried overNa₂SO₄ and filtered through a fitted funnel. Product solution assay was176.1 g (76% assay yield). This solution was carried forward into therearrangement step.

1.4 Epoxide Rearrangement and Bisulfite Formation

A solution of crude epoxide 4 (assay 59.5 g) in hexanes was solventswitched into PhMe, and added ZnBr₂ (10.7 g). When the rearrangement wascomplete judged by HPLC, the slurry was filtered through a frittedfunnel. The clear filtrate was washed with 10% aq. NaCl and then stirredwith a solution of sodium bisulfite (NaHSO₃, 24.7 g) in H₂O (140 mL)vigorously at RT for 3 hr. The cloudy aqueous layer was separated andwashed with heptanes. By ¹H-NMR assay, the aqueous solution contained71.15 g bisulfite adduct 6 (30.4 wt % solution, 90% yield from crudeepoxide 4). This solution was used directly in the subsequenttransaminase step.

1.5 Transaminase DKR

To a cylindrical Labfors reactor was charged pyridoxal-5-phosphate (1.4g, 5.66 mmol), 452 ml 0.2 M borate buffer pH 10.5 containing 1M iPrNH₂,52 g transaminase (SEQ ID NO: 180), and 75 ml DMSO, and the resultingmixture was warmed to 45° C. The pH was controlled at pH 10.5 using 8 Maq iPrNH₂. To this was added dropwise a mixture of 17.16 wt % aqsolution of ester bi-sulfite 6 (147.2 g, 353 mmol) and 219 ml DMSO underN₂ atmosphere. When the reaction was complete judged by HPLC, thereaction mixture was cooled and extracted with 1 volume of 3:2 IPA:IPAc.The aq/rag layer was extracted again with 1 volume of 3:7 IPA:IPAc. Theorganic layer was washed with brine at pH >9. Assay yield in solutionwas 78 g (87%); 99.3% ee. After dried over MgSO₄, and filtered through afitted funnel, the crude solution was concentrated under vacuum flushingwith IPAc to remove IPA. The resulting slurry was concentrated to afinal volume of ˜200 mL, cool to below 0° C., and filtered to collectthe solid. The cake was washed with ice-cold IPAc and dried at RT undervacuum to give the desired product (84% corrected yield, 99.3 LCAP) as awhite powder.

Alternative Substrates

While the DKR transaminase reaction described above was performed usingthe bisulfite adduct of ester aldehyde 5, it was also determined thataldehyde 5 itself is a good substrate for DKR transamination using theprocess as described above.

When screening two transaminases, SEQ ID NO: 18 and SEQ ID NO: 180,using either the ester aldehyde 5 of its bisulfite adduct 6, theenantiomeric excess of each reaction was found to be 99% or greater.

1.6. Reduction of Amide

The lactam 7 can be reduced to form the piperidine 8 as described below:

A mixture of lactam 7 (10.25 g at 97.5 wt %) in THF (100 mL) was cooledto <10° C., and added NaBH₄ (4.47 g). EtOH (6.89 mL) was then addedslowly over 20 min. The slurry was aged for an additional 1 hr at 2° C.after which BF₃.THF (13.03 mL) was added over 1 hr. The slurry wasslowly warmed to RT and aged until complete conversion judged by HPLC.The reaction was then cooled to <5° C. then slowly quenched with MeOH(7.96 mL), added HCl (9.69 mL), then the reaction was heated to 45° C.until decomplexation of product-borane complex was complete, asindicated by LC assay. The reaction was cooled, diluted with IPAc (75mL) and water (80 mL), and then pH was adjusted with aqueous NH₄OH to pH8. The organic layer was separated, added 75 mL water, then pH adjustedto 10.5 with 50 wt % NaOH. The layers were separated and the organiclayer was washed with brine. After solvent-switched to IPAc, LC Assayyield was 9.1 g; 95.9%.

Example 2

The DKR transaminase reaction described in Example 1 can be performedusing related substates, yielding similar products. For example, thefollowing schemes can also be used to generate the key intermediatedescribed as part of the present invention.

Example 3

Different transaminases were tested in the reaction as described inExample 1 (section 1.5), using the ester aldehyde 5 as the substrate toform the lactam 7 by DKR transamination.

Pryridoxal-5-phosphate (1 mg; 4.05 μmol) was dissolved in water 0.2Mborate buffer pH 10.5 (400 μl) containing isopropylamine hydrochloride(50 mg, 0.523 mmol). The respective transaminase (2 mg), 0.663 mmol) wasadded and slowly dissolved. Then a solution of ester aldehyde 5 (2 mg,6.39 μmol) in DMSO (100 μl) was added and the reactions aged at 45° C.As control, ester adehyde 5 (2 mg, 6.39 μmol) was incubated in water0.2M borate buffer pH 10.5 (400 μl) and DMSO (100 μl) with and withoutthe isopropylamine hydrochloride (50 mg, 0.523 mmol).

The enantiomeric excess was determined for the transaminases tested inthis reaction, the results shown in the table below:

Transaminase Sequence % e.e. 1 SEQ ID NO: 18 99 2 SEQ ID NO: 207 75 3SEQ ID NO: 208 59 4 SEQ ID NO: 209 66 5 SEQ ID NO: 210 44 6 SEQ ID NO:211 45

What is claimed is:
 1. A process for preparing an asymmetric compound ofFormula I:

wherein: R¹ is a leaving group, a protected amino group, NO₂, or OH orits protected form; R² is hydrogen; R³ is (C═O)OR⁵, CH₂R⁶, or aprotected aldehyde; or, R² and R³ are combined to form a nitrogencontaining heterocyclyl selected from

R⁴ is hydrogen or an amino protecting group; R⁵ is C₁₋₆ alkyl, C₃₋₁₀cycloalkyl, C₄₋₁₀ heterocyclyl, aryl, or heteroaryl; and, R⁶ is aleaving group or OH or its protected form; comprising a biocatalytictransamination of a compound of Formula II:

wherein: R¹ is as defined above; R²′ is an aldehyde or an aldehydeequivalent; and, R³′ is R³; or R²′ and R³′ are combined to form

in the presence of a transaminase polypeptide, a coenzyme, and an aminodonor.
 2. The process of claim 1, wherein the biocatalytictransamination provides a compound of Formula I having an enantiomericexcess of at least 95%.
 3. The process of claim 2, wherein thetransaminase polypeptide is selected from SEQ ID NO: 18 or SEQ ID NO:180.
 4. The process of claim 3, wherein the transaminase polypeptide isSEQ ID NO:
 180. 5. The process of claim 1, wherein the coenzyme ispyridoxal-phosphate (PLP)
 6. The process of claim 1, wherein the aminodonor is isopropylamine.
 7. The process of claim 1, wherein thetransaminase polypeptide is SEQ ID NO: 180, the coenzyme is PLP, and theamino donor is isopropylamine.
 8. The process of claim 1, wherein R¹ isBr.
 9. The process of claim 1, wherein R² and R³ are combined to form anitrogen containing heterocyclyl selected from

and R⁴ is hydrogen.
 10. The process of claim 9, wherein R² and R³ arecombined to form

and R⁴ is hydrogen.
 11. The process of claim 1, wherein R¹ is Br, R² andR³ are combined to form

and R⁴ is hydrogen.
 12. The process of claim 1, wherein R² is hydrogen,R³ is CH₂R⁵, R⁴ is hydrogen, and R⁵ is OH.
 13. A process for preparingan asymmetric compound of Formula III:

comprising a biocatalytic transamination of a compound of Formula IV ora compound of Formula V:

in the presence of a transaminase polypeptide, a coenzyme, and an aminodonor.
 14. The process of claim 13, wherein the biocatalytictransamination provides the compound of Formula III having anenantiomeric excess of at least 95%.
 15. The process of claim 14,wherein the transaminase polypeptide is selected from SEQ ID NO: 18 orSEQ ID NO:
 180. 16. The process of claim 15, wherein the transaminasepolypeptide is SEQ ID NO: 180, the coenzyme is PLP, and the amino donoris isopropylamine.
 17. A process form preparing an asymmetric compoundof Formula VI:

comprising: (a) a biocatalytic transamination of a compound of FormulaIV or a compound of Formula V:

in the presence of a transaminase polypeptide, a coenzyme, and an aminodonor, forming the compound of Formula III:

(b) reducing the lactam of the compound of Formula III, forming thecompound of Formula VII:

(c) protecting the piperidine nitrogen of the compound of Formula VII toform the compound of formula VI.